Adipocytes were long considered inert components of the bone marrow niche, but mouse and human models suggest bone marrow adipose tissue (BMAT) is dynamic and responsive to hormonal and nutrient cues.In this study of healthy volunteers, we investigated how BMAT responds to acute nutrient changes, including analyses of endocrine determinants and paracrine factors from marrow aspirates. Study participants underwent a 10-day high-calorie protocol, followed by a 10-day fast.We demonstrate (a) vertebral BMAT increased significantly during high-calorie feeding and fasting, suggesting BMAT may have different functions in states of caloric excess compared with caloric deprivation; (b) ghrelin, which decreased in response to high-calorie feeding and fasting, was inversely associated with changes in BMAT; and (c) in response to high-calorie feeding, resistin levels in the marrow sera, but not the circulation, rose significantly. In addition, TNF-α expression in marrow adipocytes increased with high-calorie feeding and decreased upon fasting.High-calorie feeding, but not fasting, induces an immune response in bone marrow similar to what has been reported in peripheral adipose tissue. Understanding the immunomodulatory regulators in the marrow may provide further insight into the homeostatic function of this unique adipose tissue depot.NIH grant R24 DK084970, Harvard Catalyst/The Harvard Clinical and Translational Science Center , and NIH grants P30 DK040561 and U19 AG060917S1. Bone marrow adipose tissue (BMAT) is a major component of the bone marrow microenvironment, yet its function is not well understood. Although individuals with anorexia nervosa, a psychiatric illness characterized by self-induced starvation and low body weight and a model of chronic starvation, have low levels of subcutaneous (SAT) and visceral adipose tissue (VAT) , BMAT leThe rapidity of energy changes appears to affect BMAT changes. We previously demonstrated that women with a history of anorexia nervosa who are now at normal body weight have lower levels of BMAT compared with low-weight women with active anorexia nervosa . In contWe hypothesized that acute nutrient changes would cause rapid changes in BMAT and that these changes would be associated with hormones and immune modulators associated with increased caloric intake, starvation, and/or body weight. We therefore investigated BMAT changes in humans in response to a structured, acute, short-term weight gain protocol followed by an acute, short-term weight loss protocol. We measured BMAT changes in response to these acute changes in body weight and nutritional status and investigated the hormonal and inflammatory predictors of these changes.n = 10 women) were admitted to the Translational and Clinical Research Center (TCRC) at the Massachusetts General Hospital for a 10-day inpatient high-calorie visit. The goal of this portion of the study was to achieve a 7% weight gain during the 10-day admission. Subjects were subsequently discharged for approximately 2 weeks, during which time they were instructed to resume their normal diet, and then readmitted to the TCRC for a 7- to 10-day inpatient fast, during which time subjects were not allowed any caloric intake. Baseline characteristics of the study participants are listed in 1H-magnetic resonance spectroscopy (1H-MRS) with a primary endpoint of change in BMAT at the L4 vertebra, femoral diaphysis and femoral metaphysis based on our prior work demonstrating differences in BMAT at these 2 sites in women with anorexia nervosa compared to normal-weight controls (P = 0.02), femoral diaphysis (P = 0.002), and femoral metaphysis (P = 0.004) was significantly greater in men at baseline as compared with women.Twenty-three subjects  and the femoral metaphysis . There was also a significant inverse association between BMAT at the femoral diaphysis and SAT ; in contrast to these prior data, there was an inverse association between adiponectin and BMAT at the femoral metaphysis  and femoral metaphysis . In female subjects, there was also a positive association between SAT and BMAT at the L4 vertebra , in contrast to what we previously observed in a population of normal-weight and low-weight, premenopausal women , consistent with data in a rodent model demonstrating increased bone marrow adiposity in response to infusion with ghrelin , VAT , trunk fat , extremity fat , and lean mass  all increased during the high-calorie visit  (P = 0.08), but this increase was not statistically significant ; there were no other significant associations between changes in BMAT and changes in body composition parameters during the high-calorie visit. Although the baseline BMI range of our study subjects was narrow . There were no other associations observed between baseline BMI and changes in BMAT during the high-calorie visit.BMAT at the L4 vertebra also increased significantly during the high-calorie visit  . There wnificant . CorrelaP = 0.02), suggesting that greater increases in SAT during the high-calorie visit were associated with lesser increases in BMAT at the femoral metaphysis during the 2-week stabilization period. Similarly, in men, percentage change in SAT during the high-calorie visit was inversely associated with percentage change in L4 BMAT during the 2-week stabilization period , again suggesting that the greater the increase in SAT with high-calorie feeding, the greater the decrease in L4 BMAT in the 2 weeks after the high-calorie visit (2-week stabilization period). In contrast, in men, percentage change in VAT during the high-calorie visit was positively associated with percentage change in BMAT at the femoral metaphysis during the 2-week stabilization period , potentially suggesting a very different relationship between BMAT and VAT as compared with BMAT and SAT in response to high-calorie intake. In men, there was also a significant positive association between percentage change in lean mass and percentage change in BMAT at the femoral diaphysis in response to high-calorie feeding .Comparing changes in the men and women during the high-calorie visit, there were no significant differences in changes in weight, SAT, VAT, or BMAT at any site between men and women , whereasP < 0.0001; P < 0.0001), trunk fat , extremity fat , and lean mass  decreased significantly , whereas women gained significantly more BMAT at the femoral metaphysis . Women, who had gained more lean mass than men during the high-calorie visit, similarly lost significantly more lean mass during the fasting visit as compared with men  during the fasting visit. Therefore, the greater the drop in weight with fasting, the greater the increase in BMAT at the femoral metaphysis in women. In contrast to what was observed in men in response to high-calorie feeding (a positive association between change in BMAT and change in lean mass), percentage change in L4 BMAT was inversely associated with change in lean mass in women  with fasting. This difference could potentially be due to (a) a difference in the association between BMAT and lean mass between the sexes, (b) a difference in the association between BMAT and lean mass in the setting of nutrient sufficiency versus nutrient deficiency, or (c) a difference in the association between BMAT at a more proximal location (L4 vertebra) versus a more peripheral BMAT depot . Changes in VAT and SAT during the fasting visit were not correlated with changes in BMAT during the fasting visit in either men or women.In women, there was a strong inverse association between percentage change in weight and percentage change in BMAT at the femoral metaphysis (P < 0.0001) during the 2-week stabilization period, during which participants were instructed to resume their normal diet at home after the high-calorie visit (P = 0.19), but VAT decreased a median of –11.4%  . Similar to the baseline correlation, percentage change in lean mass was positively associated with percentage change in L4 BMAT . There were no other significant associations between changes in body composition and changes in BMAT during the 2-week stabilization period, but percentage change in L4 BMAT during this period was positively associated with baseline BMI . Baseline BMI was not associated with changes in femoral BMAT during this stabilization period.There were significant decreases in BMAT at the L4 vertebra and femoral diaphysis during the 2-week stabilization period . BMAT atP = 0.001; R = –0.58, P = 0.005) percentage change in BMAT at the femoral diaphysis during the 2-week stabilization period .Changes in BMAT during the 2-week stabilization period were significantly associated with changes in BMAT during the high-calorie visit and/or changes in BMAT during the fasting visit. Percentage change in BMAT at the L4 vertebra during the 2-week stabilization period was inversely associated with percentage change in BMAT at the L4 vertebra during the high-calorie visit . Baseline BMI was also significantly and positively associated with percentage change in L4 BMAT in both women  and men .When comparing changes in women versus men, there were no significant differences with respect to change in BMAT, SAT, VAT, weight, or body composition parameters as measured by DXA during the 2-week stabilization period . ChangesP < 0.0001), adiponectin (P < 0.0001), and IGF-1 (P < 0.0001) all increased significantly during the high-calorie visit, whereas ghrelin, an orexigenic hormone, decreased by a mean of –28.9% ± 4.7% (P < 0.0001) and IGF-BP2 decreased by a median of –41.3%  (P < 0.0001) (P = 0.002), whereas there was no significant change in P1NP (P = 0.75), a marker of bone formation (P = 0.0005), and CRP increased a median of 135.4%  (P = 0.002).Leptin ( 0.0001) . With reormation . InflammP = 0.002; P = 0.04; As part of an exploratory analysis to determine hormonal predictors of BMAT, we investigated univariate associations between changes in BMAT with high-calorie feeding and changes in adipokines, bone turnover markers, inflammatory markers, and changes in G-CSF levels, given recent data demonstrating suppression of marrow adipocytes by G-CSF in a murine model ref. . We founP < 0.0001) and adiponectin (P < 0.0001) decreased significantly in response to fasting. This drop in adiponectin is consistent with prior human data demonstrating a decrease in adiponectin levels with fasting (P < 0.0001). With respect to bone turnover markers, CTX increased  and P1NP decreased  significantly in response to fasting. CRP, which increased in response to high-calorie feeding, also increased in response to fasting , and GDF15, another inflammatory marker, also increased in response to fasting . In contrast to the observed baseline association in female study participants, there was a positive association between percentage change in FGF21 and percentage change in BMAT at the femoral diaphysis . Consistent with data in a murine model demonstrating suppression of marrow adipocytes in response to endogenous G-CSF  from the same individual undergoing 1 of the 2 nutritional challenges but not both. For marrow serum markers, we assessed adiponectin, IL-6, and resistin levels paired before and after the nutritional intervention. Peripheral blood adiponectin levels increased after 10 days of the high-calorie diet and decreased significantly after the fasting intervention (P < 0.05), but this rise was not reflected in peripheral blood levels (data not shown). In addition, there was a nonsignificant decline in marrow sera resistin 10 days after fasting but again no change in peripheral blood resistin with fasting. IL-6 did not change in the marrow sera of individuals undergoing the high-calorie or fasting intervention (data not shown).We performed whole bone marrow aspirations on 9 subjects at baseline and on the final day of the high-calorie visit and 11 distinct participants on the baseline day and final day of the fasting visit. This led to paired samples  have been performed. These studies demonstrate that BMAT, which has been shown to represent approximately 13% of total adipose tissue in healthy individuals , is inveWe have previously shown that BMAT appears to behave differently in states of chronic starvation as compared with states of nutritional sufficiency. For example, although BMAT is positively associated with VAT in studies including overweight/obese women  and as wIntriguingly, the largest change in BMAT was not during high-calorie intake or during fasting but instead during the 2-week period when subjects resumed their normal diet after high-calorie feeding. During this 2-week period, there was a 17.8% drop in L4 vertebral BMAT in women and a 20.4% drop in L4 vertebral BMAT in men. Whether this drop in BMAT reflects a redistribution of lipid stores after a period of acute weight gain warrants further study. Our data also support the concept of region-specific variation in BMAT response to environmental cues. We have previously demonstrated in murine models that distal skeletal BMAT sites are less responsive to systemic challenges compared with proximal skeletal BMAT sites . Here weData from marrow aspirates in this study provide a unique perspective on the adipocytic response to nutrient changes. High-calorie feeding induced a proinflammatory response with increases in TNF-α, Plexin D1, and SEMA3E gene expression in mature adipocytes at day 10, as well as a marked increase in marrow serum resistin, a putative measure of macrophage activation. These proinflammatory mediators induce the recruitment and activation of adipose tissue macrophages (ATMs). The activated ATMs secrete proinflammatory cytokines and form the inflammatory circuit, which blocks the insulin action of adipocytes and leads to insulin resistance. On the other hand, no markers of inflammation were noted in the fasting aspirates after 10 days . Taken tThe timing of caloric excess or deprivation (chronic vs. acute) plays a major role in hormonal responses. We demonstrate that although ghrelin levels decreased in response to high-calorie feeding, as would be expected, given ghrelin’s role as an orexigenic hormone, ghrelin levels also decreased significantly with fasting. This unexpected finding contrasts with what is observed in a state of chronic caloric deprivation, anorexia nervosa, in which ghrelin levels are elevated –24. One In murine models, FGF21, like ghrelin, is a hormonal determinant of BMAT. FGF21 is a hormone induced during starvation in murine models as well as in humans and is a mediator of ketogenesis in murine models but not in humans , 25, 26.In conclusion, we demonstrate in an acute 10-day high-calorie feeding protocol followed by a 10-day fasting protocol that BMAT increased in response to both interventions, but the pathophysiology and cues for these changes likely differ. These data support the concept that nutrient flux rather than body weight is an important determinant of BMAT. A limitation of our study is that the fasting intervention followed the high-calorie protocol for all study subjects. Therefore, changes that we observed during fasting were likely in part modified by changes during an acute high-calorie intervention. Nevertheless, these data demonstrating changes in body composition, appetite-regulating hormones, bone turnover markers, and inflammatory markers after a high-calorie intervention and a fasting intervention will provide a resource for future human studies. This work also demonstrated a number of observations in humans, including the remarkable changes in BMAT with acute changes in nutrient intake, as well as the significant decrease in ghrelin after both a high-calorie intervention as well as a fasting intervention. Finally, bone marrow aspirates during acute nutrient fluxes, which have not been reported previously to our knowledge, revealed a remarkable difference in the marrow environment in response to a high-calorie diet compared with fasting.We studied 23 individuals (10 women and 13 men). All individuals (mean age and age range: 33.3 years and 22–44 years) were normal weight or overweight (BMI range: 23.3–27.9). No subject had a history of diabetes mellitus or a history of an eating disorder, and none of the participants were taking chronic medications. All women were premenopausal and had a history of regular menstrual cycles, and none had used exogenous estrogen within 3 months of her baseline visit. An additional female participant enrolled in the study (age at enrollment: 31 years and BMI at enrollment: 24.8) but was not able to comply with the high-calorie protocol and did not gain weight during the first study visit ; therefore, her data have not been included in the analyses.n = 6 men and n = 3 women) underwent bone marrow aspiration (described below) at baseline and on the final day of the high-calorie visit, and 11 subjects  who did not undergo bone marrow aspiration during the high-calorie visit had a bone marrow aspiration on both the baseline day and final day of the fasting visit.All subjects were admitted to the TCRC at the Massachusetts General Hospital for 2 inpatient study visits totaling up to 20 days. Participants were initially admitted for a 10-day high-calorie visit, during which time they met with a bionutritionist, who calculated their caloric needs for 7% weight gain based on body weight using the Mifflin St Jeor equation with an activity factor of 1.3 , 29. ParAll subjects underwent DXA on the baseline day of each inpatient visit and on the final day of the high-calorie visit and the final fasting day. Body composition parameters including trunk fat, extremity fat, and lean mass were measured by DXA. Coefficients of variation (CV) for DXA have been reported to be no more than 1.1% for total body lean mass and range from 2.4% to 6.7% for trunk fat and extremity fat measurements .A single axial magnetic resonance imaging  slice (at the level of the L4 vertebra) was used to measure abdominal SAT and VAT.1H-MRS was performed to determine lipid content in the marrow . For the L4 vertebra, a voxel measuring 15 × 15 × 15 mm (3.4 mL) was placed within the L4 vertebral body. Single-voxel 1H-MRS data were acquired using point-resolved spatially localized spectroscopy (PRESS) pulse sequence without water suppression with the following parameters: echo time of 30 ms, repetition time of 3000 ms, 8 acquisitions, 1024 data points, and receiver bandwidth of 2000 Hz. For the femur, a voxel measuring 12 × 12 × 12 mm (1.7 mL) was positioned within the metaphysis at the inter-trochanteric region, and single-voxel 1H-MRS using the same non–water-suppressed PRESS pulse sequence was performed. This process was repeated with voxel placement in the mid-diaphysis. Automated procedures for optimization of gradient shimming and transmit and receive gain were used. We have reported the CV for marrow fat quantification in a population of premenopausal women in our institution to be 3% (1H-MRS data was performed using LCModel software (version 6.1-4A) (Stephen Provencher). Data were transferred from the scanner to a Linux workstation, and metabolite quantification was performed using eddy current correction and water scaling. A customized fitting algorithm for bone marrow analysis provided estimates for all lipid signals combined . LCModel bone marrow lipid estimates were automatically scaled to unsuppressed water peak (4.7 ppm) and expressed as lipid/water ratio. 1H-MRS assessment of the L4 vertebra for a subject before and after both the high-calorie feeding and fasting visits is illustrated in Fitting of the g for 8 minutes at 4°C. The top layer was then transferred to a microfuge tube; TRIzol reagent was added  and the samples were stored at –80°C. Samples in the tiger-top tubes were spun at 2054g for 10 minutes at 4°C. The top layer was aliquoted and stored at –80°C. The majority of cells were mature adipocytes, but the possibility that a small number of adherent stromal vascular cells were isolated with floated adipocytes cannot be excluded.Subjects were placed in the prone position, and the posterior iliac crest was sterilely prepared using the ChloraPrep applicator (CareFusion). Two percent lidocaine was used to infiltrate the subcutaneous tissues and periosteum. Using an 11G OnControl bone marrow biopsy device , a bone marrow aspirate was obtained using standard protocols  and collSerum leptin, adiponectin, FGF21, G-CSF, IL-6, TNF-α, resistin, and GDF15 were measured by human DuoSet ELISA . For leptin, the intra-assay CV was 3.2% and the inter-assay CV: 4.4%. The intra-assay CV for adiponectin was 3.9% and the inter-assay CV: 6.5%. For FGF21, the intra-assay CV was 3.4% and the inter-assay CV: 7.5%. The intra-assay CV for G-CSF was 1.9% with an inter-assay CV of 3.7%. For IL-6, the intra-assay CV was 2.6% and the inter-assay CV: 4.5%. For TNF-α, the intra-assay CV was 2.0% and the inter-assay CV was 6.5%. The intra-assay CV for resistin was 5.0%, The intra- and inter-assay CV for resistin were less than 9.2%. The intra-assay CV for GDF15 was 2.3% with an inter-assay CV of 5.4%. Ghrelin was measured by ELISA (Thermo Fisher Scientific) with an intra-assay CV of 6.0% and an inter-assay CV of 8.5%. P1NP, a marker of bone formation, and CTX, a marker of bone resorption, were measured by a luminescent immunoassay analyzer . For P1NP, the intra-assay CV was 3.0% and the inter-assay CV was 5.0%. For CTX, the intra-assay CV was 3.2% and the inter-assay CV was 6.2%. IGF-BP2 was measured by ELISA (ALPCO) with a detection limit of 0.2 ng/mL, and intra- and inter-assay CV were both less than 10%. IGF-1 was measured in a clinical laboratory (Quest Diagnostics Nichols Institute). Insulin and CRP were also measured by a clinical laboratory (Quest Diagnostics), and glucose was measured as previously described . HOMA-IRg for 8 minutes at 4°C. The remaining components were divided into 3 compartments: compartment 1, pellet, which contained the stromal vascular fraction; compartment 2, the fluid between the pellet and the top layer, which we called marrow serum because of its appearance; and compartment 3, the top layer of floated cells. Measurements of adiponectin, IL-6, and resistin were performed on marrow sera using the conventional sandwich ELISA kits  according to the manufacturer’s instructions. The detection limits were 1 ng/mL, 5.0 pg/mL, and 10 pg/mL, respectively.After the bone marrow aspiration, PBS 5 cc was added to the aspirate  and collected in the EDTA tube. Bone marrow aspirate was then spun at 377Total RNA was purified from mature adipocytes using the RNeasy Micro Kit, which included a DNase I treatment step as per manufacturer’s instructions (QIAGEN). First-strand cDNA was synthesized from 0.5 μg total RNA using the iScript cDNA Synthesis Kit (Bio-Rad). Primer sequences used for real-time quantitative PCR are listed in t test. Non-normally distributed data were reported as median [interquartile range] and were compared using the Wilcoxon test. Paired-sample 2-tailed t test or Wilcoxon signed-rank test  was used to compare changes in body composition parameters and hormone levels between the baseline day and final day of each study visit and the final day of the high-calorie visit and the first day of the fasting visit (to assess changes during the 2-week stabilization period). For normally distributed data, Pearson’s correlation coefficients (represented by R) were calculated to assess univariate relationships. For non-normally distributed data, Spearman’s coefficients (represented by rho) were calculated to assess univariate relationships. A P value of less than 0.05 was considered significant.Statistical analysis was performed using JMP Pro 13.0 (SAS Institute) software. Normally distributed data were reported as means ± SEM and compared using the 2-tailed The study was approved by the Partners HealthCare Institutional Review Board  and complied with the Health Insurance Portability and Accountability Act guidelines. Written informed consent was obtained from all subjects.PKF, MAB, HL, OAM, MCH, CJR, and AK participated in the study design. PKF, MAB, WZ, XZ, ATF, MR, SPP, TMH, HL, and EKO conducted the human experiments and/or were responsible for acquisition of data and their analysis. GPP and CJR were responsible for the design and conduct of the bone marrow aspirate analyses. PKF and CJR contributed to the writing of the manuscript. All authors critically reviewed and approved the manuscript.


Evidently, there is room for the development of new types of PECH-C-based AEMs with great potential for energy generation in the RED process.The development of the most effective, suitable and economic ion-exchange membranes is crucial for reverse electrodialysis (RED)—the most widely studied process to harvest salinity gradient energy from mixing seawater and river water. RED utilizes two types of membranes as core elements, namely cation exchange membranes (CEM) and anion exchange membranes (AEM). Since the preparation of AEMs is more complex compared to CEMs, the design and development of anion exchange membranes have been the focus in this study. Homogeneous AEMs based on two types of polyepichlorohydrin (PECH) with different chlorine amounts  were synthesized, and first-time benchmarking of the membrane properties was conducted. In addition to physicochemical membrane properties, some instrumental analyses such as SEM, FTIR and DSC were investigated to characterize these anion-exchange membranes. Based on the results, although the PECH-H-type membrane had enhanced ion-exchange properties, PECH-C-based anion-exchange membranes exhibited a higher power density of 0.316 W/m Energy, water and food have represented three global issues faced by humankind for a long time. In particular, energy consumption and the search for alternative energy sources have become inevitable due to the depletion of fossil fuels and undesired climate change. One of the most interesting and probably the least-known renewable energy source is osmotic energy ). This type of energy, also called blue energy, has great potential worldwide since it is the sustainable energy extracted from the mixing of two water bodies with different salinities, such as river water and sea water ,2,3,4,5.There are now many techniques being developed to extract salinity-gradient energy and convert it into electrical energy, such as pressure-retarded osmosis (PRO), reverse electrodialysis (RED), and capacitive mixing (CAPMIX), which are commonly known ,7,8. HowRED is an electrochemical process in which the ion-exchange membranes are used as core elements. It is basically the inverse of conventional electrodialysis (ED) ,21, suchPolyepichlorohydrin (PECH)-based AEMs have recently gained much attention because of single-step fabrication without the use of toxic chloromethylation reaction. In addition, simultaneous quaternization and crosslinking is possible when tertiary diamines are used to prepare these AEMs. Since the tailor-made AEMs for RED were first introduced in 2012 by Güler et al. , there hIn this work, two types of PECH (polyepichlorohydrin (PECH-H) and polyepichlorohydrin-co-ethylene oxide (PECH-C))-based AEMs were particularly prepared for RED applications. For the first time, PECH-C-based AEMs were fabricated as promising alternatives as RED membranes. Simultaneous crosslinking and functionalization were performed without the use of toxic chloromethylation step. In particular, the chemical composition of AEMs synthesized and the correlations with the physicochemical properties were investigated. First-time benchmarking of these properties with the commercially available membrane has been also performed using the lab-scale RED system.2Cl)CH2O)m(CH2CH2O)n, EPICHLOMER C, 25 wt% chlorine and ((CH(CH2Cl)CH2O)n H, 37 wt% Cl, Osaka Soda, Co., Ltd., Osaka, Japan), polyacrylonitrile, Mitsubishi Chemical Co. Ltd., Tokyo, Japan), 1,4-diazabicyclo [2.2.2] octane  were adopted. Dimethyl sulfoxide (Isolab) was used as the solvent for all the experiments.To fabricate the homogeneous AEMs, polyepichlorohydrin ((CH(CH3[Fe(CN)6]) and potassium ferrocyanide (K4[Fe(CN)6]·3H2O) were provided from Proanalyst, Merck, Darmstadt, Germany. All chemicals were used as they were purchased from the suppliers.Sodium sulfate, silver nitrate, and sodium chloride used for membrane characterization were obtained from Isolab  Potassium ferricyanide (KAnion-exchange membranes (AEMs) with homogeneous bulk structures were fabricated by solution-casting. This was later followed by solvent evaporation as reported in our previous work . The amiIn order to prepare different sets of membranes with different compositions, two terms, namely the blend ratio (BR) and excess diamine ratio (EDR), were defined to investigate the effects of concentration of active polymer PECH with respect to inert polymer PAN, and the concentration of diamine DABCO with respect to chloromethyl functional groups in active-polymer PECH, respectively .After the removal of surface water of the PECH membrane using blotting paper, the film thickness was immediately measured using a precise micrometer  at various regions of the membrane, and the average thickness was determined.To investigate the morphology of membrane films, QUANTA 400F Field Emission SEM  was used. Membrane samples were placed on the holders and coated with gold to provide conductivity. The surface and cross-section of the membrane samples were investigated.−1 at wavelengths between 4000 and 400 cm−1.The surface characterization of the prepared dry PECH membrane samples were performed by Fourier transform infrared (FTIR) spectroscopy . Attenuated total reflectance (ATR) mode was used during the analyses. The average of 32 scans was taken for the spectrum with a resolution of 4 cm2SO4 for 3 h. To calculate the IEC, Na2SO4 solution was back-titrated with a 0.1 M AgNO3 solution [3 solution, respectively, while The ion exchange capacity (IEC) is defined as the amount of fixed-charge groups per unit weight of dry polymer. Initially, membranes were immersed in 3 M NaCl solution for 15 h at room temperature (20 °C). Then, Milli-Q water was used to rinse membranes. After that, the membranes were exposed to 1.5 M Nasolution . Lastly,The swelling degree (SD) is a physicochemical parameter that directly determines the mechanical properties of a membrane. To determine the SD, prepared anion-exchange membranes were soaked into demineralized water for 24 h. After weighing the wet membranes, they were dried until a constant weight was reached at approximately 30 °C. After measuring the dry membranes, the swelling degree was calculated using Equation (2):fix), which can be defined as the amount of ion exchanging functional groups per water content in the membrane, has an impact on the ion transport properties of the membranes. Cfix can be calculated using the ratio of IEC and SD values of the prepared membranes as defined in Equation (3) [The fixed charge density (Ction (3) .(3)Cfixm) and heat of fusion ((ΔHfusion) of the quaternized and pristine membranes were measured using Perkin Elmer (Diamond DSC) differential scanning calorimetry in the temperature range of −70 to 200 °C at a scanning rate of 20°/min under a N2 atmosphere.To observe the morphology of different PECH-type polymers and their crystallinity behavior, differential scanning calorimetry (DSC) analyses were performed. The melting temperature (T4Fe(CN)6 and 0.05 M K3Fe(CN)6) at 300 mL/min. Feed waters  were fed to the stack with peristaltic pumps at several flow rates between 30 and 120 mL/min at room temperature (20 °C). A schematic flow diagram of the lab-scale RED system is given in Custom-made AEMs (PECH-C and PECH-H) were fabricated to be used in the lab-scale RED system with a 10 cm × 10 cm electrode area . The RED stack was formed by installing three PECH-based AEMs and four Neosepta CMX cation-exchange membranes alternatingly mounted between a reversible anode and cathode . The intermembrane thickness was kept fixed at 400 µm using Nitex polyamide woven spacers. An electrode rinse solution was circulated containing the mixture of three solutions  is evaluated by dividing this power output by the total membrane area.The RED performance of the produced AEMs was investigated using a potentiostat  in chronopotentiometry mode. Up to 40 A/mIn order to attain sufficient film-forming properties and to make benchmarking with commercial AEMs, PECH-based membranes with about 150 µm were fabricated in this work. Many commercial ion-exchange membranes for RED, ED, EDI, or other electromembrane processes have about a 100–150 µm film thickness , although a few have thicker  and some have thinner film thicknesses  .To examine the anion-exchange membranes’ morphology on both the surface and cross-section, SEM analysis was performed at a magnification of 2000×. In the RED system, membranes with a dense bulk structure are preferred to prevent or reduce the water transport through the membranes. According to the SEM images, both the surface and cross-section morphology of PECH-H- and –C-type membranes are very similar . Even atTo verify quaternization  of the active PECH polymer with DABCO, FTIR analysis was performed. −1 and 2872 cm−1. At wavenumbers of 1425 cm−1 and 1440 cm−1, scissoring bands of –CH2 groups were observed. The wavenumber 743 represents C-Cl bondings of the functional groups of PECH polymers. On the other hand, a -C≡N bond of pristine PAN was visible at peak 2240 cm−1. The same peak was still visible at the blend of PECH and PAN polymers. The peak at 3400 cm−1 represented O-H stretches, indicating free water. When the active polymer is aminated with DABCO, C-N bonds appeared at 1640 cm−1, showing successful quaternization of all membranes. Nevertheless, the quaternized PECH-C-type membrane had more intense peaks, which can be estimated to have a relatively higher amount of functional groups [In l groups ,35. In aDifferently from our previous work, the effects of PAN-PECH blending on membrane properties were investigated at various blend ratios between 0.6 and 2. In addition, to determine the optimized PECH-based AEM for RED application, two types of PECH were employed: one has a homopolymer of epichlorohydrin, (PECH-H) and the other has the copolymers of epichlorohydrin and ethylene oxide, (PECH-C). The effect of blending PECH and PAN on IEC and SD for the prepared membranes was shown in For both types of PECH membranes, IEC and SD increased with an increasing blend ratio. High values of IEC and SD were observed up to 4.0 mmol/g and 140%, respectively. These results are in the same direction with the data reported by Güler et al. , where sAccording to The effect of excess diamine ratio (EDR) on SD and IEC is investigated between 1.2 and 4.0 . In our In As the polymer structures of PECH-H- and -C-type membranes are different, this can be confirmed by investigating the morphology by differential scanning calorimetry (DSC) analyses. The impact of differences in the alignment of molecular chains  on the ion-exchange capability of the membranes can also be investigated by these analyses.fusion), melting temperature (Tm), chain flexibility and interactions. These factors also have a significant impact on the mechanical properties [There are some vital parameters affecting the crystal structure of the homogenous membranes such as enthalpy of fusion  and ΔHfusion (3.9104 J/g) of PECH-C than Tm (154.48 °C) and ΔHfusion (2.9835 J/g) of PECH-H, PECH-C-type membranes have more a crystalline character . Thus, anion  transport through the membrane is reduced, and it yields a low ion-exchange capacity.In DSC analysis, both crystalline and semi-crystalline polymers melt when heated. Highly crystalline polymers require more energy to break or vibrate the polymeric chains. Therefore, an interpretation of the direct correlation between the melting point and crystallization can be made . Owing tharacter . CrystalTo summarize the properties of the prepared membranes and make benchmarking with commercial counterparts, the data are shown in 2. It is worth mentioning that it is possible to make these membranes as thin as 100 µm and even thinner according to our past experience. By ensuring this, we expect a superior RED performance compared to other commercially available alternatives.Here, for the first time, we report the performance of ethylene-oxide containing PECH anion-exchange membranes (PECH-C) in the RED system. Their performance is compared to commercial counterparts. All the membranes were prepared with a similar film thickness, having a BR of 0.6 and EDR of 2.0 . The RED2, whereas it was 0.39 W/m2 for Neosepta AMX coupled with Neosepta CMX at the same operating conditions. However, there is always some room for development of PECH-based membranes, which will be always an attractive option for RED processes because of its single-step fabrication without toxic reactions such as chloromethylation.For the first time, we have shown a comparison between PECH-based membranes containing ethylene oxide copolymer (PECH-C) and the other membranes based on RED performance. These membranes were made via a single-step quaternization-crosslinking method, which represents a highly promising alternative for custom-made RED membranes. The highest power density produced by PECH-C-type membranes coupled with Neosepta CMX was 0.32 W/m


No study has revealed the effect of porcine brain enzyme hydrolysate (PBEH) on memory impairment. We aimed to examine the hypothesis that PBEH intake modulates memory deficits and cognitive behavior in scopolamine (SC)-induced amnesia rats, and its mechanism, including gut microbiota changes, was determined. Sprague–Dawley male rats had intraperitoneal injections of SC (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), PBEH  at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day through a feeding needle for six weeks. The Normal-control rats had casein feeding without SC injection. PBEH dose-dependently protected against memory deficits determined by passive avoidance test, Y-maze, water-maze, and novel object recognition test in SC-induced rats compared to the MD-control. The High-PBEH group had a similar memory function to the Positive-control group. Systemic insulin resistance determined by HOMA-IR was lower in the PBEH groups than in the Normal-control but not the Positive-control. In parallel with systemic insulin resistance, decreased cholesterol and increased glycogen contents in the hippocampus in the Medium-PBEH and High-PBEH represented reduced brain insulin resistance. PBEH intake prevented the increment of serum TNF-α and IL-1β concentrations in the SC-injected rats. Hippocampal lipid peroxide and TNF-α contents and mRNA TNF-α and IL-1β expression were dose-dependently reduced in PBEH and Positive-control. PBEH decreased the hippocampal acetylcholinesterase activity compared to the MD-control, but not as much as the Positive-control. PBEH intake increased the α-diversity of the gut microbiota compared to the MD-control, and the gut microbiota community was separated from MD-control. In metagenome function analysis, PBEH increased the energy metabolism-related pathways of the gut microbiota, including citric acid cycle, oxidative phosphorylation, glycolysis, and amino acid metabolism, which were lower in the MD-control than the Normal-control. In conclusion, alleviated memory deficit by PBEH was associated potentially with not only reducing acetylcholinesterase activity but also improving brain insulin resistance and neuroinflammation potentially through modulating gut microbiota. PBEH intake  can be a potential therapeutic agent for improving memory impairment. The prevalence of dementia due to Alzheimer’s disease and cerebral stroke is rising as many countries become aging societies. Although the cause of the neurodegenerative disease is unknown, it is associated with the amyloid-β plaque and neurofibrillary tangles by tau hyperphosphorylation and neurotransmission disturbance . ElevateScopolamine is a non-selective post-synaptic muscarinic receptor blocker that occupies the muscarinic receptor with high affinity and activates acetylcholinesterase in the cortex and hippocampus . It alsoConsiderable research has been conducted to investigate the natural products and prevent or slow the progression of amnesia and Alzheimer’s-type dementia . Most stOn the other hand, no study has examined the ability of porcine brain enzyme hydrolysate (PBEH) by protease in a scopolamine-induced animal model to inhibit the acetylcholine activity and modulate the gut–brain axis. This study hypothesized that the PBEH supplementation modulated memory deficit and abnormal cognitive behavior in the amnesia animal model and was associated with gut microbiota changes. The hypothesis was examined in scopolamine-induced amnesia rats, and the potential mechanism of PBEH was also revealed. This study reports a potential functional food that protects against memory impairment.Total nitrogen contents of PBEH contained 6.95 ± 0.56 mg/mL, and its peptide contents (2–5 kd) were 65%. PBEH mainly contained L-arginine (2.36 ± 0.55 mg/mL), L-leucine (2.31 ± 0.42 mg/mL), and L-phenylalanine (1.61 ± 0.17 mg/mL) .The scopolamine intraperitoneal injection-induced memory deficit was measured by passive avoidance, Y-maze, novel object recognition tests for short-term memory, and water-maze test for spatial memory. In the second trial of the passive avoidance test, the Normal-control had a higher latency time than the control, and only the High-PBEH extended the time A. In theIn the water-maze test, latency time to the first visit in zone 5, where the platform was placed, was longer in the MD-control than the Normal-control, while the donepezil and High-PBEH treatments reduced the latency time to find zone 5 compared to the Normal-control. The frequency and duration to visit zone 5 were lower in the MD-control than the Normal-control, whereas donepezil and PBEH supplementation prevented the decrease as much as the Normal-control C.Scopolamine-developed depression was indicated by decreasing retaining swimming rate, which was much lower in the MD-control than the Normal-control . Positivp < 0.05; p < 0.05; Memory deficit can be modified with systemic and brain insulin resistance, which may change energy and glucose metabolism. However, final body weight and weight gains during six weeks were not significantly different among the MD-control, Positive-control, and Normal-control groups, while they lowered in the Medium-PBEH and High-PBEH groups compared to the MD-control , the serum glucose concentrations increased until 30–40 min, and they decreased in all rats A. The seMemory deficit is also associated with neuroinflammation interacted with systemic inflammation. The serum tumor necrosis factor-α (TNF-α) concentrations were higher in the MD-control than in the Normal-control, but they decreased in the Medium-PBEH, High-PBEH, and Positive-control groups . The serThe hippocampal insulin resistance, oxidative stress, and neuroinflammation are associated with memory deficits. Brain triglyceride, cholesterol, and glycogen contents indirectly represent brain insulin resistance. The hippocampal triglyceride contents were not significantly different among the groups but tended to be lower in the Medium-PEBH than in the MD-control . On the Hippocampal lipid peroxide contents, indicating brain oxidative stress, were higher in the MD-control than the Normal-control, and donepezil and PBEH lowered the contents. The High-PBEH group showed a decrease to as low as the Normal-control . The aceTNF-α and IL-1β was higher in the MD-control than the Normal-control, and High-PBEH lowered them as much as the Normal-control  was lower in the MD-control than the Normal-control, while Positive-control and High-PBEH increased it to a similar level to the Normal-control (CNTF) mRNA expression showed a similar pattern to BDNF, but its expression was lower in the MD-control than the Normal-control. The increase by PBEH and donepezil did not reach the Normal-control , glycolysis, oxidative phosphorylation, and some amino acid degradation compared to the MD-control. It might be related to lower weight gain in PBEH groups than MD-control. The present study suggested that PEBH could be a potential functional food to protect against memory impairment.BDNF expression in the hippocampus by inhibiting the acetylcholinesterase activity and reducing proinflammatory cytokines in the present study. These changes by PBEH were associated with reversing the memory impairment by scopolamine injection.Scopolamine is a non-selective post-synaptic muscarinic receptor blocker to activate acetylcholinesterase to impair cholinergic neurotransmission . The modDonepezil is a potent acetylcholinesterase inhibitor, and the US FDA approved donepezil for dementia treatment, but its long-term efficacy and safety are unclear. Diet interventions such as protein hydrolysates have been studied for dementia prevention . In the Alzheimer’s disease is associated with decreased glucose utilization in the brain related to insulin resistance . DonepezLactobacillus and Bifidobacterium species is related to neurodegenerative diseases [Lactobacillus, Blautia, and Dorea decreased, and Biophilia and Bacteriodes increased in the Medium- and High-PBEH as much as the Normal-control.Neurodegenerative diseases can be prevented by changing the daily diet to modulate the microbiota–gut–brain axis . The gutdiseases . PBEH inInterestingly, their changes caused a significant alteration in metagenomic function determined by Picrust2. The gut microbiome indicated that energy metabolism-related pathways differed in the MD-control and Normal-control. However, MD-control and Normal-control had similar body weight, and it indicated that scopolamine altered energy metabolism and gut microbiota changes compensated it. By contrast, PBEH intake in MD rats decreased the body weight and visceral fat mass compared to the MD-control without decreasing the food intake. These results suggest that energy expenditure might be elevated in PBEH, but it is not applied to MD-control and Normal-control. The metagenome data suggested that the Medium- and High-PBEH intake elevated glycolysis, citric acid cycle, β-oxidation, and some amino acid degradation compared to the MD-control. The increment in the PBEH groups was similar to the Normal-control. Therefore, the body weight changes might be related directly to the gut microbiota changes in a scopolamine injection, and PBEH intake reversed its changes. In addition, the gut–brain axis influences memory function and weight regulation .In conclusion, a long-term scopolamine injection induced memory dysfunction, and PBEH intake  protected against its induction as much as the donepezil treatment. Although PBEH and donepezil inhibited acetylcholinesterase activity in the brain, PBEH was a weaker inhibitor than donepezil. However, PBEH improved insulin resistance and reduced the lipid peroxide and IL-1β contents in the hippocampus better than donepezil. PBEH and donepezil did not show adverse effects, including liver damage. Therefore, with additional clinical research, PBEH can be an alternative therapy for alleviating human memory deficits. Blood was removed from a porcine brain with distilled water, and ethanol was added to dissolve fat components and precipitate proteins. The precipitates were digested with protease at 37 °C for 26 h, which was inactivated. Undigested proteins were precipitated with ethanol and filtered using the ultrafiltration method. The filtrates showed a 58% yield. The filtrates (PBEH) mainly contained 2–5 kd peptides, and they were used for the animal feeding study. As described previously, amino acid contents in PBEH were determined using an amino acid analyzer  . Peptidead libitum. All procedures conformed with the Guide for the Care and Use of Laboratory Animals (8th edition) issued by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Hoseo University (HSIACUC-17071).Seventy male Sprague–Dawley rats aged eight weeks (216 ± 13.0 g) were purchased from Daehan Bio Inc. . The rats were acclimatized for seven days in an animal facility. Each animal was housed in an individual stainless-steel cage in a controlled environment  with access to food and water Experimental design is presented in ad libitum during the entire experimental period.All rats had free access to a high-fat diet prepared using a modified polyphenol-free AIN-93 semi-purified method, which has been shown to induce insulin resistance and exacerbate memory deficits from scopolamine administration . The semA rat quickly entered the dark box in the passive avoidance apparatus equipped with a two-compartment dark/light shuttle box when left in the light shuttle box . ElectroThe Y-maze test was conducted in a horizontal Y-shaped maze with three arms, 50.5 cm in length, 20 cm in width, and 20 cm in height. A rat was located in one arm, and its movements were monitored for 8 min. Consecutive entry into all three arms of the Y-maze was considered a correct alternation . The perThe spatial memory function was evaluated using the Morris water-maze test ,33, whicAll rats were subjected to a novel object recognition test for four days. A novel object recognition test was performed according to a previous research method with slight modifications ,34. BrieEach rat had a 10 min pretest in a clear acrylic cylinder (60 cm in height and 30 cm in width) filled with up to 45 cm water at 24 ± 1 °C. After 24 h of the pretest, the 5 min swim test was performed to make a score for active (swimming and climbing) or passive (immobility) behavior when the rats were forced to swim in the cylinder. The researcher randomly and blindly selected and evaluated the video of the 5 min session and measured the time for total mobile and immobile behavior. The results were shown as the active time during the 5 min session.After an overnight fast, oral glucose administration (2 g/kg body weight) was conducted. The serum glucose concentrations were measured every 10 min for 90 min and again at 120 min using a Glucometer . The serum insulin concentrations in the fasting state were determined using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) kit . HOMA-IR was calculated as follows: serum insulin (μU) × serum glucose (mmol/L)/22.5.n = 4) were fixed in the 4% paraformaldehyde. The feces in the cecum were collected and frozen at −70 °C. The next day after the water-maze test, all rats were deprived of food for 16 h to make metabolic parameters of the experimental animals not affected by previous food intake. Blood was drawn from the portal vein, the inferior vena cava, and organs were collected rapidly after anesthetizing with the mixture of ketamine and xylazine (100 and 10 mg/kg body weight). The epididymal and retroperitoneal fat masses were weighed. Six brains selected randomly from each group were frozen at −70 °C, while the rest  assay kit  and cholesterol kit (Asan Pharmaceutics). The supernatants were deproteinized with 1.5N perchloric acid, and the glycogen content was calculated from the glucose concentrations derived from glycogen hydrolyzed by α-amyloglucosidase in an acid buffer [The hippocampi were divided into two sections: one part was lysed with RIPA buffer containing protease inhibitors, and the other was randomly selected to be lysed with Trizol reagent  for extracting the total RNA. The hippocampal RIPA buffer lysates were centrifuged at 5000× d buffer . The glud buffer . Triglycv:v:v) using polymerase chain reaction (PCR). The cDNA was mixed with the primers for the genes of interest and SYBR Green mix to determine the expressions of the designated genes using a real-time PCR machine . As previously published, the primers used for the CNTF, BDNF, TNF-α, IL-1β, and β-actin were given [cDNA was produced using a mixture of isolated total RNA from the hippocampus with superscript III reverse transcriptase and high-fidelity Taq DNA polymerase , and 1N HCl (100:1) was added to the mixture. The mixture was vortexed and centrifuged at 15,000 rpm, 15 min, and 4 °C. The SCFA concentrations in supernatants were measured by gas chromatography  using an Elite-FFAP 30 m × 0.25 mm × 0.25 μm capillary column with helium as the carrier gas at a flow rate of 1 mL/min, as described previously . ExogenoThe fecal microbiome communities were investigated using the feces from ceca by metagenome sequencing using next-generation sequencing procedures . AccordiThe 16S amplicon sequences were processed using Mothur v.1.36. MiSeq SOP was used to identify fecal bacterial taxonomy, and bacterial counts were conducted on each fecal sample. The sequences were aligned using Silva reference alignment v. 12,350, and bacteria counts and identifications for all taxa were determined, as described previously ,41. The https://www.genome.jp/kegg/tool/map_pathway1.html, accessed on 11 November 2021) [The metabolic functions of the gut microbiota were predicted from the fasta files and count tables of fecal bacteria using PICRUSt2 . The meter 2021) . The gutp < 0.05.The statistical analysis was performed using SAS version 7 . A sample size of 10 per group was determined using the G power program (power = 0.85 and effect size = 0.50) to test the main effects. The results are expressed as the means ± standard deviations (SDs). Univariate analysis was used to check the normal distribution of variables, and one-way analysis of variance (ANOVA) was used to compare the significance of the groups when the variables were normally distributed. Multiple comparisons were conducted using a Tukey test when one-way ANOVA showed a significant intergroup difference. Statistical significance was accepted for


Previously, we found that basal corticosterone pulsatility significantly impacts the vulnerability for developing post-traumatic stress disorder (PTSD). Rats that exhibited PTSD-phenotype were characterized by blunted basal corticosterone pulsatility amplitude and a blunted corticosterone response to a stressor. This study sought to identify the mechanisms underlining both the loss of pulsatility and differences in downstream responses. Serial blood samples were collected manually via jugular vein cannula at 10-min intervals to evaluate suppression of corticosterone following methylprednisolone administration. The rats were exposed to predator scent stress (PSS) after 24 h, and behavioral responses were assessed 7 days post-exposure for retrospective classification into behavioral response groups. Brains were harvested for measurements of the glucocorticoid receptor, mineralocorticoid receptor, FK506-binding protein-51 and arginine vasopressin in specific brain regions to assess changes in hypothalamus–pituitary–adrenal axis (HPA) regulating factors. Methylprednisolone produced greater suppression of corticosterone in the PTSD-phenotype group. During the suppression, the PTSD-phenotype rats showed a significantly more pronounced pulsatile activity. In addition, the PTSD-phenotype group showed distinct changes in the ventral and dorsal CA1, dentate gyrus as well as in the paraventricular nucleus and supra-optic nucleus. These results demonstrate a pre-trauma vulnerability state that is characterized by an over-reactivity of the HPA and changes in its regulating factors. Glucocorticoid (GC) hormones are secreted to the bloodstream by the adrenal gland and, upon a stressful event, play a role in orchestrating the physiological and behavioral reactions essential for the restoration of homeostasis after emotional or physical stress ,2. As suUnder unstressed conditions, GC hormones are secreted in a highly dynamic fashion with a characteristic circadian pattern of secretion of corticosterone. In addition to a circadian release, there is a distinct pattern of a rapid ultradian corticosterone secretion which consists of a near-hourly pulse frequency ,14,15. TOur group has initiated a series of studies using a well-validated animal model for PTSD that examine the role of GCs in susceptibility to extreme disruption of behavior in response to stress  ,21,22. TThe goal of this study was to identify the mechanisms that underlie both the loss of pulsatility and the differences in downstream responses. We thus examined if the PTSD phenotype is associated with HPA axis sensitivity. We examined the feedback inhibition (suppression of corticosterone following dexamethasone administration) and other modulating factors of glucocorticoid signaling dynamics such as glucocorticoid receptor (GR), mineralocorticoid receptor (MR), FK506-binding protein 51 (FKBP5), and arginine vasopressin (AVP) levels in the hippocampus.The experimental design used here is schematically depicted in n = 4/21) fulfilled criteria for PTSD-phenotype (EBR); 38% (n = 8/21) for MBR; and 43% for PBR (n = 9/21).A broad range of variations in behavioral responses was observed in PSS-exposed animals, and several subgroups were thus identified . Accordip < 0.015, p < 0.025, p < 0.03, p < 0.02, p < 0.04, and p < 0.05, respectively) and compared to PBR rats 60 and 70 min after methylprednisolone administration   = 17.2, p < 0.0001, Groups-Time Interaction: F = 2.15, p < 0.015). A nonlinear  fitting was carried out at the six time points following corticosterone peak concentrations and indicated significantly different decay constants (t1) between groups, such that EBR rats exhibited a slower decay in corticosterone levels than MBR and PBR rats  and 20 min after methylprednisolone injection, at 10 min intervals over a period of 90 min . At basectively) A .20 min post-MPST: All rats that exhibited PTSD-phenotype (4/4) demonstrated hyper-suppression (51–90%) of corticosterone in response to methylprednisolone. In contrast, most rats in the PBR and MBR behavior groups suppressed corticosterone to less than 50% of pre-methylprednisolone levels .40 min post-MPST: Most rats that exhibited PTSD-phenotype (3/4) suppressed corticosterone to more than 90% of pre-methylprednisolone levels, compared with 11.11% of the PBR group (1/9) and compared with 12.5% of the MBR group (1/8)  at the 60 min post-MPST. 70 min post-MPST: All rats that exhibited PTSD-phenotype (4/4) suppressed corticosterone to more than 90% of pre-methylprednisolone levels, compared with 55.55% of the PBR group (5/9) and compared with 12.5% of the MBR group . The Fisher LSD post hoc test confirmed a significantly more prominent pulsatile activity in the EBR group as compared with the MBR group (p < 0.015) and the PBR group (p < 0.04); differences between the EBR and MBR groups were also significant for the Bonferroni post hoc test (p < 0.035). In the recovery phase, no significant changes were found between groups though a trend towards higher amplitudes in the EBR group was still apparent   = 2.5073, p = 0.11). A technical limitation in the duration of sampling made frequency analysis futile.We next assessed the characteristics of corticosterone pulses, which were identified using the Hierarchically AdaPtive algorithm . CharactAll rats in Experiment 2 were decapitated one day following behavioral assessments , and brap < 0.004), a significant effect on the hippocampal axis  = 198, p < 0.0001) and on the hippocampal-axis interaction  = 9.9, p < 0.0001). Bonferroni post hoc tests confirmed significantly higher GR-positive cells in the nuclear layer in the dorsal CA1 areas in EBR as compared with unexposed control (p < 0.002) and MBR (p < 0.04) rats. In contrast, significantly lower GR-positive cells were found in the ventral CA1 areas in EBR compared with unexposed control (p < 0.05) and PBR (p < 0.007) rats. In all groups, the expression of GR-positive cells in the nucleus was significantly higher in the dorsal part of the CA1 than in the ventral part . In the DG (p < 0.0001) and a group–axis interaction effect  = 15.85, p < 0.0001). No effects were observed for groups, but there was a clear trend showing higher GR-positive cells in the PSS-exposed groups  = 2.6, p = 0.059). Bonferroni post hoc tests confirmed significantly higher GR-positive nuclear translocation cells in the dorsal DG areas in EBR rats compared with unexposed controls (p < 0.003). Significantly lower GR-positive nuclear translocation cells were found in the ventral DG areas in EBR compared with unexposed control (p < 0.0004), PBR (p < 0.0001), and MBR (p < 0.009) rats. In the EBR group, the expression of GR-positive cells in the nucleus was significantly higher in the dorsal part of the DG than in the ventral part (p < 0.0001), this was not true for other groups. There were significant differences in GR levels among the groups in the PVN (p < 0.00075). Significantly lower GR-positive cells were found in the PVN area in the EBR group than in the unexposed control (p < 0.02) and MBR (p < 0.0025) rats. PBR rats also exhibited significantly lower GR-positive cells compared to the MBR group (p < 0.015).As the negative feedback loop of the HPA axis is mediated via GR and MR, we assessed the dynamic response of GR and MR to PSS in the dorsal and ventral hippocampus by quantified MR and GR nuclear translocation patterns. Looking at GR-ir cells in the hippocampal CA1 subregion A, two-wan the DG B, two-wa the PVN C (One-wap < 0.02), and a groups–axis interaction effect  = 16.4, p < 0.0001). No effects were observed for groups. Bonferroni post hoc tests confirmed significantly higher MR-positive cells in the nuclear layer of the dorsal CA1 areas in EBR compared with unexposed control (p < 0.03) and MBR (p < 0.00015) rats. In contrast, significantly lower MR-positive cells were found in the ventral CA1 areas in EBR compared with unexposed control (p < 0.045) and MBR (p < 0.0025) rats. In addition, in EBR and MBR rats, the expression of MR-positive cells was region-specific: EBR rats exhibited significantly higher MR-positive cells in the dorsal hippocampus compared to the ventral part (p < 0.05), whereas the MBR rats exhibited significantly higher MR-positive cells in the ventral hippocampus compared to the dorsal part (p < 0.0001).As noted above, hippocampal MR is also thought to play a principal role in the negative feedback effects of glucocorticoids, primarily on the basal HPA tone at both the trough and peak of the diurnal cycle . In the p < 0.045), a significant effect on the hippocampus axis  = 13.0, p < 0.0007) and a significant group–axis interaction  = 27.2, p < 0.0001) on the nuclear translocation profile of MR protein. Bonferroni post hoc tests confirmed significantly higher MR-positive cells in the dorsal DG areas in EBR compared with unexposed control (p < 0.0007), PBR (p < 0.02), and MBR (p < 0.0015) rats. In contrast, significantly lower MR-positive cells were found in the ventral DG areas in EBR compared with unexposed control (p < 0.0001) and MBR (p < 0.0001) rats. In addition, in EBR and MBR rats, the expression of MR-positive cells was region-specific: EBR rats exhibited significantly higher MR-positive cells in the dorsal hippocampus compared to the ventral part (p < 0.0001), whereas the MBR rats exhibited significantly higher MR-positive cells in the ventral hippocampus compared to the dorsal part (p < 0.0001). In the PVN   = 6.8, p < 0.0015).In the DG B, a two- the PVN C, no sigfkbp5 gene) regulates HPA negative feedback by preventing nuclear translocation of the GR complex . Therefore, we examined the protein levels of FKBP5 in the cytosolic fraction of ventral, dorsal hippocampus, and PVN. In the CA1  = 26.8, p < 0.0001, respectively], and a group–axis interaction effect . No effects were observed for groups. Bonferroni post hoc tests confirmed significantly lower FKBP5-positive cells in the dorsal CA1 and DG subregions in EBR compared with unexposed control , MBR  and PBR  rats. Furthermore, significantly higher FKBP5-positive cells were found in the ventral CA1 subregion in EBR rats compared with unexposed controls (p < 0.05). In addition, significantly lower FKBP5-positive cells were found in EBR rats in the dorsal subregions compared with ventral parts . In the PVN (p < 0.0025)  = 5.7, p < 0.004).The FK506-binding protein-51 , MBR (p < 0.0001) and PBR (p < 0.0001) rats. Significant differences in AVP levels were noted between groups in the SON (p < 0.0001]. Bonferroni post hoc tests confirmed significantly higher AVP- positive cells in the SON in EBR rats compared with unexposed controls (p < 0.0001), PBR (p < 0.0001) and MBR (p < 0.0001) rats. We next quantified magnocellular and parvocellular cells in the PVN separately (See methods). Significant differences in AVP in magnocellular cells were noted between groups in the PVN  = 7.6203, p = 0.00082). Bonferroni post hoc tests confirmed significantly higher AVP-positive magnocellular cells (p < 0.025), MBR (p < 0.0001) and PBR (p < 0.025) rats. Lesser, but still significant, differences in AVP were also found in parvocellular cells between groups in the PVN . Bonferroni post hoc tests confirmed significantly higher AVP-positive parvocellular cells (p < 0.045) rats. These findings indicate alterations in the hypothalamic expression of AVP only in PTSD-phenotype rats in the SON and in the PVN in both the parvocellular and magnocellular neurosecretory systems.Since AVP has long been recognized as a neuropeptide and involved in both the modulation of HPA axis activity  and negae fibers A in the  the SON B [F presented a complete blunting of pulsatile patterns during the suppression phase, i.e., no amplitudes were detected. This was not true in the PBR group where fewer than half of the rats presented a pulse during the suppression phase (4/9 44%); these pulses were presented as “escape pulses,” with quick rises of short duration. In the EBR group, pulsatile activity was seen in all but one of the rats (3/4 75%). Average amplitudes of the EBR group were also higher in size compared to those of the PBR group . During These results are intriguing. Taken together with our previous results, a clearer picture of the over-reactivity of the HPA axis as a contributing factor to the development of PTSD is starting to emerge. Although we were not able to test corticosterone levels at the onset of PSS, prominent feedback inhibition of corticosterone release is still evident in the PTSD-phenotype rats by the pre-trauma MPST response, although only partly. In addition, there is an implication of a more prominent forward-driving regulation of corticosterone release in rats later exhibiting a PTSD phenotype, evident by pulsatile activity during MPST and by the blunting of baseline amplitudes without a change in the mean baseline in our first experiment.The dysregulation of the HPA axis is extensively studied in PTSD research. In a recent review , the mosOur results strongly support that the HPA-dysregulation, found in PTSD, is a pre-trauma vulnerability trait. Furthermore, it points to a dysregulation that is indeed feedback-dependent and composed of both a negative inhibition of the axis and a positive forward drive.The positive drive of the HPA axis arising from the PVN is usually thought of as a tonic drive that is propagated by inputs from the SCN and other regions and can be either augmented by stress or blunted by feedback inhibition ,51,52,53We next analyzed the harvested brains to assess the consequences of PSS exposure on GR and MR signaling in the hippocampus and hypothalamic PVN . We founIt has been suggested that the dorsal hippocampus and ventral hippocampus play distinct functional roles; this segregation of functions along the axis of the hippocampus appears at several levels of the local network organization including principal cell properties , synaptiTo interpret our results, we first focused on the hippocampal areas that regulate the PVN and HPA axis to “trace back” the fault in regulatory mechanisms of the HPA axis activity. The ventral hippocampus, which corresponds to the anterior part of hippocampus in humans , is invoWhile it is tempting to leave it at that, this would be an over-simplification as there are several other factors to keep in mind. First, while the differences in HPA regulation were seen prior to trauma, the differences in GR and MR expression seen in the PTSD-phenotype group were measured 8 days post trauma. It is well documented that aversive events can lead to changes in receptors expression ,96,97,98Our findings of reduced MR and GR in the PVN support previous findings by Louvart et al. , and mayOne such mechanism, contributing to receptors of glucocorticoid’s sensitivity, is a large chaperone protein complex, FKBP5. When FKBP5 is bound to the GR via HSP90, the receptor has a lower affinity for its ligand and is retained in the cytoplasm ,104. WhiNext, we assessed AVP; while AVP is known to play a role in propagating brain neurotoxicity in traumatic brain injury and cerebral edema ,118,119,The involvement of the vasopressinergic system in the PSS-induced behavioral and HPA axis disruptions was evaluated 8 days after exposure by examining the levels of AVP-positive cells and fibers in the PVN and SON hypothalamus nuclei. We found that the PTSD-phenotype was associated with a pattern of persistent and excessive expression of AVP in the PVN and SON nuclei, the sites in which AVP gene expression and synthesis are strongly affected by glucocorticoids ,131,132.Surprisingly, over-expression of AVP in the PTSD-phenotype group was evident in both the magno- and parvo-cellular cell groups; these are two separate neurosecretory systems, with different physiological functions, morphology, origins, electrophysiology, and projections ,139,140.It is important to emphasize that the dysregulation in basal and reactive HPA axis in PTSD is complex and may also involve a variety of steroid compounds, including the neuroactive steroids, such as dehydroepiandrosterone (DHEA), its sulphate ester dehydroepiandrosterone sulphate (DHEA-S), allopregnanolone and pregnanolone. Alterations in the synthesis of neuroactive steroids has been associated with increased PTSD symptoms in humans ,142,143 n = 21) resulting in a small number of EBR rats  and its regulating factors as predictors and/or origins of PTSD development.Our study is not devoted of limitations in establishing the exact interaction, this owing to measuring the regulating factors at different time points, due to the nature of brain immunohistochemistry technique. Another limitation is that the PSS-stress response was not measured thus we can only correlate pre-trauma suppression patterns to behavioral outcomes and not the stress reaction itself, furthermore serial blood samples during MPST were not taken for a long enough period to establish a clear pulsatile pattern. An additional limitation of this study is the relatively small sample size of the MPST experiment  in comparison with MBR (resilient group) rats. Moreover, they suggest a role for increased GR and MR availability in the dorsal hippocampus and reduced MR and GR availability in the ventral hippocampus in the development of PTSD-like behavioral responses by stressed rats . These findings fit well with previous results obtained by our research group and support the value of their PTSD model. The most significant findings of this study are: (1) Over-sensitivity of the HPA axis, as evident by hyper-suppression of the HPA axis in response to MPST, suggests a pre-trauma vulnerability factor for traumatic exposure, which may predict the subsequent development of PTSD. (2) This pre-trauma vulnerability is characterized not only by enhanced feedback inhibition of the HPA axis but by an overall over-regulation of this axis, including an over-reactiveness of forward-driving mechanisms as seen through the prism of the increased size and number of amplitudes in conjunction with hyper-suppression during an MPST. (3) PTSD phenotype rats exhibited long-term regionally distinct alteration of the signaling of GR-MR-FKBP5 in the hippocampus and in the hypothalamic nucleus in response to PSS, attenuated GR and MR, complemented by elevation in FKBP5, expression in the ventral hippocampus and PVN, and augmented GR and MR complimented by a decrease in the FKBP5 expression in the dorsal hippocampus. This is important for glucocorticoid feedback inhibition of the HPA axis and stress signaling cascade, as well as other mechanisms of fear extinction and memory consolidation. (4) PTSD phenotype rats exhibited specific changes in the patterns of AVP expression in the hypothalamic nuclei PVN and SON. (5) Not all exposed rats subsequently developed PTSD phenotype, and not all pre-existing vulnerability factors/risk factors determined the individual’s likelihood of developing PTSD phenotype upon stress exposure. (6) The association between risk factors and diseases is complex and multifactorial, influenced by a variety of factors involving genetic risk and environmental conditions interfacing with a variety of potentially interacting “hits” and vulnerability factors occurring during exposure, contrary to the binary “two hit model” that was proposed nearly 30 years ago.All procedures were performed under strict compliance with ethical principles and guidelines of the NIH Guide for the Care and Use of Laboratory Animals. All treatment and testing procedures were approved by the Animal Care Committee of the Ben-Gurion University of the Negev, Israel 18 December 2018 (IL-06-09-2018).A total of 30 adult male Sprague-Dawley rats, weighing 190–320 g (21 rats underwent MPST and 9 rats for control), were used in this study. The rats were housed (three per cage) in a vivarium with a stable temperature under a 12:12-h light-dark cycle , with unlimited access to food and water. All rats were allowed a 1-week habituation period before the experiment. All procedures were performed during the resting phase of the rats between 08:30 and 12:00. After jugular vein catheter surgery, rats were housed singly in opaque plastic bins with restricted access to one another through a mesh barrier. The mesh barrier cages consist of a 40 cm × 28 cm × 20 cm high transparent Perspex box Each chamber contained a removable partition that separated the chamber into two equal-sized compartments. The mesh barrier was made of wire grid mesh (1.5 cm × 1.5 cm), allowing the rats to insert their noses into the adjacent compartment. Rats were handled daily following jugular catheterization.This study aimed at assessing the strength of negative feedback sensitivity of the HPA axis and regulating factors. We used methylprednisolone suppression tests (MPSTs), which were performed 7 days following the post-surgical recovery period. Serial blood samples were collected manually via a jugular vein cannula in conscious rats at 10-min intervals to evaluate the suppression of corticosterone following methylprednisolone  (945 µg/kg). After 24 h, rats were exposed to PSS or sham-PSS. Behaviors were assessed on day 7 using the EPM and the ASR paradigms. The prevalence rates of extreme, partial, and minimal behavioral responses  were assessed. One day later, rats were sacrificed, and their brains were collected for an immunoreactivity analysis of GR-ir, MR-ir, AVP-ir, and FKBP52-ir in hippocampus subregions and hypothalamic nuclei.Our study focused on brain regions essential for glucocorticoid feedback inhibition of the HPA axis and stress signaling cascade, including the PVN and dorsal and ventral hippocampus. The hypothalamic nuclei and hippocampus were chosen as targets in this study because these two regions appear to be key sites for glucocorticoid feedback inhibition of the HPA axis ,28,85. WRats were individually placed on well-soiled cat litter, which was used by a cat for 2 days and sifted for stools. The rats were exposed to the litter for 10 min in a plastic cage (inescapable exposure) placed on a yard paving stone in a closed environment ,23,160. The behavior of rats was assessed in the EPM and ASR paradigms, as described previously ,23,160 aThe classification of individuals according to the degree to which their behavior was affected by a stressor is based on the premise that extremely compromised behavior in response to the priming trigger is not conducive to survival and is thus inadequate and maladaptive at representing a pathological degree of response. The severity of the response of each rat to the stressor is classified as either an extreme behavioral response (EBR), a partial behavioral response (PBR), or a minimal behavioral response (MBR) ,23,160 . All rats that underwent jugular catheterization surgery had to be isolated to prevent tampering with the cannula and surgical stitches. To prevent isolation anxiety, rats were handled daily and put in housing that was separated by a mesh barrier as described elsewhere  and xylazine (6 mg/kg) i.p. The right jugular vein was exposed, and a polyurethane cannula  was inserted into the vessel until it lay close to the entrance of the right atrium. The cannula was prefilled with pyrogen-free heparinized (10 IU/mL) isotonic saline. The free end of the cannula was exteriorized through an incision between the shoulder blades and then capped. Following surgery, animals were housed in individual cages for recovery for 7 days prior to beginning the blood sampling protocol. Jugular cannulas were manually flushed daily by withdrawing 0.2 mL of blood and replacing the volume with 0.3 mL of sterile heparinized saline  to maintain patency of the cannula. For the blood sampling following recovery, the end of the capped cannula was attached to a special harness connected to a mechanical swivel via a long metal spring that rotated 360° in a horizontal plane and 180° through a vertical plane to maximize freedom of movement for the rats . For Experiment 1, blood samples were collected by hand (700 µL) every 20 min (for a duration of 11.5 h); for Experiment 2, blood samples were collected by hand (300 µL) every 10 min (for a duration of 90 min). Blood samples were stored in Eppendorf tubes. Serum was separated by centrifugation and then stored at −80 °C until processed for corticosterone measurements.Corticosterone was measured with a DSL-10-81000 ELISA kit  according to the instructions of the manufacturer by a person blind to the experimental procedures. All samples were measured in duplicates.A methylprednisolone suppression test was used to measure feedback inhibition of the HPA axis activity. Each rat received a push intravenous injection of 165 μg/kg (approx. 40–50 μg) methylprednisolone , and blood samples were collected at baseline (prior to methylprednisolone injection), and every 10 min for a duration of 90 min starting at 40 min post-methylprednisolone administration. Due to a prominent yet more rapid onset of GC suppression and faster recovery, methylprednisolone was chosen over dexamethasone for the GC suppression test, as it better fits our technical limitations of serial blood sampling ,162,163.Tissue preparation: Twenty-four hours after the behavioral tests, rats were deeply anesthetized and perfused transcardially with cold 0.9% physiological saline, followed by 4% paraformaldehyde  in a 0.1 M phosphate buffer. Brains were quickly removed, postfixed in the same fixative for 12 h at 4 °C, cryoprotected overnight (30% sucrose in 0.1 M phosphate buffer at 4 °C), and then frozen and stored at −80 °C. Serial coronal sections (10 μm) were obtained with a cryostat  and mounted on coated slides.Staining: Sections were air-dried and washed three times in phosphate-buffered saline (PBS) containing Tween 20 (PBS/T) . They were incubated for 60 min in a blocking solution  and then overnight at 4 °C with the primary antibodies against GR [rabbit polyclonal anti-GR antiserum (1:300), product code: sc-ANT-010, Alomone Labs, Jerusalem, Israel), MR , product code: sc-ANT-010, Alomone Labs, Jerusalem, Israel), AVP (Rabbit Anti-Vasopressin antibody (1:500), product code: ab68669, Abcam, Israel), FKBP5 , product code: ab2926, Abcam, Israel). After three washes in PBS/T, the sections were incubated for 2 h in DyLight-488-labeled goat-anti-rabbit IgG or in Dylight-594 goat-anti-mouse IgG  in PBS containing 2% normal goat or horse serum. The sections were subsequently washed and mounted with a mounting medium . Sections of the brains of different groups of rats were processed at the same time and under identical conditions to ensure reliable comparisons and to maintain stringency in tissue preparation and staining conditions. Control staining was performed in the absence of the primary antibodies. Additionally, secondary fluorescent labels were swapped to test for cross-reactivity, and sections were incubated without primary antibodies to test for non-specific binding of the secondary antibodies.AVP immunoreactivity was examined in parvo- and magnocellular parts of the hypothalamic PVN and in the SON. Magnocellular neurons were differentiated histologically from the parvocellular neurons based on their overall size, morphology, and location, as described in previous studies of the PVN cellular mappings ,165.http://rsb.info.nih.gov/ij/, accessed on 3 March 2021), in the same manner as described previously [To quantify differences in MR and GR subcellular distribution patterns, changes in fluorescence intensity values of nuclear immunoreactivity were measured, using ImageJ 1.32j analysis software . Corticosterone pulses analysis: All corticosterone ultradian pulsatile data were analyzed using an adaptation of Hierarchically AdaPtive analysis, an algorithm written as a Matlab-based code, designed for analysis of hormonal pulsatility, created by Dean et al. [Data are presented as the mean ± SEM unless otherwise specified. n et al. . The ampp < 0.05 was considered statistically significant.For the GR, MR, and FKBP5 levels, statistical analyses were performed with a two-way ANOVA with groups  and hippocampus axis  as the independent factors, or one-way ANOVA (for the PVN area). For the AVP levels, statistical analyses were performed with a one-way ANOVA. Bonferroni tests were used to examine differences between individual groups. To gain an additional understanding of the relationship between behavioral measures and corticosterone pulsatility parameters, two sets of regression analyses were conducted. Pearson’s correlation analysis was used to describe the relationship between each behavioral variable  and corticosterone pulsatility parameters across the entire sample. The contribution of each variable that exhibited a significant correlation (or trend) was then evaluated using a multiple-stepwise regression analysis. An alpha level of


Mechanical properties of powder bed fusion processed unalloyed copper are reported majorly in the as-fabricated condition, and the effect of post-processes, common to additive manufacturing, is not well documented. In this study, mechanical properties of unalloyed copper processed by electron beam powder bed fusion are characterized via room temperature quasi-static uniaxial tensile test and Vickers microhardness. Tensile samples were extracted both perpendicular and parallel to the build direction and assigned to three different conditions: as-fabricated, hot isostatic pressing (HIP), and vacuum annealing. In the as-fabricated condition, the highest UTS and lowest elongation were obtained in the samples oriented perpendicular to the build direction. These were observed to have clear trends between sample orientation caused primarily by the interdependencies between the epitaxial columnar grain morphology and dislocation movement during the tensile test. Texture was insignificant in the as-fabricated condition, and its effect on the mechanical properties was outweighed by the orientation anisotropy. The fractographs revealed a ductile mode of failure with varying dimple sizes where more shallow and finely spaced dimples were observed in the samples oriented perpendicular to the build direction. EDS maps reveal that grain boundary oxides coalesce and grow in HIP and vacuum-annealed specimens which are seen inside the ductile dimples and contribute to their increased ductility. Overall, for the post-process parameters chosen in this study, HIP was observed to slightly increase the sample’s density while vacuum annealing reduced the oxygen content in the specimens. The benefits of complex geometries, reduced assemblies, and rapid build time attributed to powder bed fusion (PBF) additive manufacturing (AM) have recently been extended to unalloyed copper for a variety of applications requiring very high thermal and electrical conductivity, complicated geometries, and extensive processing routes. Examples include, but are not limited to, electric motors , vacuum In part, the lack of component-level data can be attributed to the significant processing challenges associated with PBF-AM of copper. Recent data from the literature suggest that the feasible AM process window for both laser PBF (L-PBF) and electron beam PBF (EB-PBF) is effectively narrow and highly sensitive to thermal boundary conditions and geometric influences within a layer. These effects are exacerbated by the high thermal conductivity of the material ,14. Ther−3–2 × 10−6 mBar) contributes toward minimizing oxygen pickup, and the high beam power and scan speeds facilitate efficient bed preheating to reduce global thermal gradients [For L-PBF, the high thermal conductivity, high reflectance , and oxidation are well documented and often cited as the most significant process barriers for fabrication ,15. Receradients ,25, althAs expected, the reported ultimate tensile strength (UTS) and yield data are skewed by sample density. This is notably evident in the L-PBF studies, where the reported density has a wide range. Lykov et al.  reportedApart from powder bed fusion AM processes, Kumar et al. have reported UTS values of 176.4 ± 6.5 MPa with 67.2 ± 2.2% elongation for copper produced using a binder jet (BJ) AM that was sintered in a reducing atmosphere and subsequently subjected to hot isostatic pressing (HIP) at 1075 °C . While oAlongside the solid density, the effect of oxygen content on ductility, particularly outside the range of what would typically be considered pure copper, is well documented . Myers aNitrogen gas-atomized copper powder screened to −270/+635 mesh was obtained from Sandvik Osprey . The initial oxygen content of the powder was 457 wt. ppm, measured using inert gas fusion . Powder particle size measurement was carried out on a Microtrac S3500  using a laser diffraction technique. The unalloyed copper powder chemistry was obtained using the inductively coupled plasma mass spectroscopy  method. Powder morphology was observed using a JEOL 6010LA scanning electron microscope .−4 mbar. Five builds, each containing three rectangular blocks measuring 58.5 mm × 12.7 mm × 58.5 mm, were fabricated upon an 86 mm diameter and 40 mm thick oxygen-free copper base plate using a customized Arcam A2 EB-PBF system , the details of which are described by Ledford et al. . The nomZ when the blank was cut with the long sample axis parallel to the build direction and X when the long sample axis was perpendicular to the build direction using the ASTM nomenclature [−5 mbar (5 × 10−9 MPa). After treatment, all samples were machined into ASTM subsize specimens (specimen type 4) [aR using a stylus profilometer .nclature . Each of type 4) . The surThe grip section of each tensile bar was cut perpendicular to the longitudinal axis approximately 10 mm from each end. These samples, in turn, were sectioned longitudinally with a low-speed diamond saw . The cut face was potted in phenolic resin and progressively ground using 400, 600, 1000, 2000 and 4000 grit SiC paper with water lubricant. Samples were polished with 1-micron, 0.3-micron, and 0.05-micron alumina slurry, and etched with Ammonium Persulphate solution by submersion/swabbing for 10 s and rinsing to reveal the grain structure. Samples were indented for Vickers microhardness measurements in a LECO M400 hardness tester  under 100 gf load for a dwell time of 10 s. A total of ten indentations, 500 µm apart, were made along the direction of tension for both types of sample orientation, and the average values are reported. The sectioned grip end that was not used for metallographic analysis was used to measure the density of the sample using gas pycnometry (Quantachrome Ultrapyc 1200E according to ). ApproxThe SEM image shown in E), defined as: P is the beam power in J/s, v is the beam velocity in mm/s, h is the scan line offset in mm and t is the layer thickness in mm, the used parameter set resulted in an average volumetric energy density of 92.3 J/mm3, as shown in 3 . Specimens subjected to both vacuum-anneal heat treatment and HIP exhibited a slightly higher density  compared to as-fabricated samples (<p = 0.0001), however no significant difference in density was detected between the HIP and vacuum-annealed specimens (p = 0.084). It should also be noted that the overall densification was relatively small from the as-fabricated to treated conditions (only 0.45%), as the as-fabricated density was already 8.88 ± 0.1 g/cm3.p > 0.15), the block number from which the samples were harvested (p > 0.41). These trends are observed in The plots in p = 0.0003), HIP with 195.7 ± 2.2 and 175.3 ± 4.3 MPa (p < 0.0001), and vacuum-annealed with 195.5 ± 3.8 and 166.2 ± 17.6 MPa (p = 0.0007), respectively (see p = 0.931), respectively, after HIP 87.3 ± 9.9 and 74.5 ± 7.6 MPa (p = 0.0475), or after vacuum-annealing, 56.7 ± 9.4 and 52.2 ± 12.3 MPa (p = 0.42). The as-fabricated samples consistently had the highest yield values, averaging just over 100 MPa, but also the widest variance due most likely to residual defects such as localized unhealed layer defects. The HIP treatment had an average midrange value for yield stress, ~20% lower than the as-fabricated condition, and a reduced scatter for both orientations (p = 0.0016). Consistently low yield stress values were observed in the vacuum-annealed samples, ~46% lower than the as-fabricated condition (p < 0.0001) and ~32% lower than the HIP condition (p < 0.0001).The X-oriented specimens exhibited higher UTS compared to Z-oriented specimens for all three processing conditions: as-fabricated with 211.2 ± 14.7 and 177.2 ± 8.5 MPa (vely see A. The avp = 0.0005), 77% higher  in the HIP condition (p < 0.0001), and 56% higher  after vacuum-annealing (p = 0.0018). The elongation of the as-fabricated Z-oriented specimens approaches the expected values for annealed copper [2. Their reported as-fabricated samples had UTS of 231.6 MPa (X-oriented) and 177 MPa (Z-oriented), yield of 149.8 MPa (X-oriented) and 78 MPa (Z-oriented), and elongation ranging from 56.2–59.3%. The microhardness data in While the strength values were observed to be influenced by harvest orientation (UTS) and post-treatment condition (0.2% yield), the most obvious property affected was elongation, as shown in d copper ,39. The d copper ,12 for sFor metals fabricated by processes which tend to form columnar grains or anisotropic microstructures such as by drawing, rolling, forging, or additive manufacturing, it is generally accepted that the properties differ by orientation due to the anisotropy ,45,46,47The reconstructed EBSD IPF maps in Most of the literature reported on mechanical properties of PBF and directed energy deposition (DED) are in a narrow material space, often focusing on commercially available alloys such as Ti-6Al-4V, IN718, 304 SS, 316L SS, Co-28Cr-6Mo, among others ,45. In pmT was used in this study for HIP and vacuum-annealed samples and led to residual stress relief, thus resulting in lower yield strength and microhardness values, as shown in Since our study used unalloyed copper and samples were fabricated to high densities at temperatures exceeding 300 °C, some of the effects often reported in PBF and DED studies such as frequent layer defects, second phase precipitation, and micro-cracking become less influential. While no strong crystallographic texture was measured by EBSD in any of the X-orientation samples, columnar grains were clearly observed and may explain the differences in our mechanical properties, especially elongation and UTS. In a strain- and defect-free pure metal system, dislocation motion in the direction of long, thin grains would have more unobstructed movement in comparison with deformation across narrow grains, experiencing frequent grain boundaries. Therefore, the tensile samples harvested from the blocks in the build direction had significantly higher elongation as compared with the specimens harvested perpendicular to the build direction where fewer grain boundaries and oxides were present to impede deformation. The samples harvested in the X direction had consistently higher UTS compared with those in the Z direction due to significantly more interactions with grain boundaries and oxides during deformation, regardless of treatment. The yield was observed to be influenced by treatment conditions regardless of orientation and most likely is the result of any remaining residual stress which may resist initial deformation. These residual stresses, caused by the numerous thermo-mechanical cycles during the fabrication process, affect the bulk yield strength on a macro-scale where high yield strength values are reported for AM-processed materials with high internal residual stresses . Heating2O particles. These large-scale oxides appear on the dimples due to the stress concentration and dislocation movement around them during the deformation process, similar to what was previously illustrated in as-fabricated EB-PBF copper by Guschlbauer et al. [Fracture surfaces were observed with SEM to explore the ductile fracture. r et al.  and sintr et al. . HoweverThe STEM EDS maps in Mechanical properties of the as-fabricated specimens are observed to be similar to unalloyed annealed copper. X-oriented specimens have higher tensile strength values across post-processes than the Z-oriented samples with an average UTS of 211.2, 195.7 and 195.5 MPa in as-fabricated, HIP, and vacuum-annealing conditions, respectively. Maximum elongation to failure is recorded for Z-oriented vacuum-annealed and HIP specimens that showed average values of ~63%, as opposed to ~54% in as-fabricated samples.With the density values reaching close to the theoretical density, the observed differences in UTS and elongation are proposed to be controlled by orientation anisotropy and interdependencies between the grain morphology and dislocation movement. Difference in yield strength is more pronounced between treatment conditions than the specimen orientation  and is assumed to be affected by the differences in residual stress as a function of treatment conditions.Density of the specimens increased slightly after the HIP treatment with an associated increase in ductility and decrease in strength. As expected, the vacuum-annealing process reduced the oxygen content within the specimen, also resulting in the lowest strength among all treatment conditions.The EBSD maps show no apparent texture in the as-fabricated specimens, and a slight amount of texture in HIP and vacuum-annealed specimens. However, the effect of such texture in the mechanical properties is outweighed by morphological anisotropy due to the epitaxial columnar microstructure formed during the fabrication process.While all treatment conditions show a ductile failure mode irrespective of the sample harvesting direction, HIP and vacuum-annealed specimens are noticeably different due to the presence of large micron-scale oxide particles within the dimples. It is suggested that the high temperature used in these post-processes promoted the coalescence and growth of grain boundary oxides which are mostly in the nano-scale size in the as-fabricated condition. This bigger oxide size led to larger interparticle distances and resulted in increased void spacing that, in turn, increased ductility in the HIP and vacuum-annealed condition.In this study, the effects of different post-processing conditions on the mechanical properties of EB-PBF fabricated unalloyed copper are characterized and discussed. Rectangular blocks were fabricated with enabled automatic control settings from ~99.95% pure copper powder with a size distribution of 22–63 µm. Tensile specimens were extracted from these rectangular blocks with two different orientations and treated with HIP and vacuum-annealing alongside the as-fabricated condition. The following conclusions summarize the major findings of this study: